Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Many biomedically significant proteins, including antibodies, cytokines, anticoagulants, blood clotting factors, and others are glycoproteins. Thus, there is a high demand for systems that can be used to produce recombinant glycoproteins for basic biomedical research and direct clinical applications. Unfortunately, few currently available recombinant protein production systems can produce higher eukaryotic glycoproteins with authentic carbohydrate side chains. Furthermore, no currently available system can produce large amounts of recombinant glycoproteins in properly glycosylated form at relatively low cost.
The glycoproteins are a major subclass of proteins distinguished by the presence of oligosaccharide side chains, or glycans, covalently linked to the polypeptide backbone. It has been estimated that over half of the proteins encoded by the human genome are modified by glycosylation and this subclass includes many different types of biomedically significant proteins, as noted above. Furthermore, glycoprotein glycans are much more than mere chemical decorations of the polypeptide backbone. In fact, glycoprotein glycans have been implicated in a wide variety of important biochemical and biological functions, including protein stability, trafficking, serum half-life, immune function, enzymatic function, cellular adhesion, and others.
Influenza viruses are enveloped, single stranded RNA viruses with two type I membrane glycoproteins, hemagglutinin (HA) and neuraminidase (NA), projecting from the virion surface. Each year, seasonal influenza affects 3-5 million people worldwide and causes 300,000-500,000 fatalities. A pandemic outbreak of a new influenza virus strain, like the one that occurred in 1918, could kill tens of millions of people worldwide. Vaccination is the most effective means of preventing influenza infections. Antibodies induced against HA's are protective; thus, HA content is used to standardize influenza vaccines. Seasonal influenza vaccines consist of three strains of influenza, including two strains of influenza A and one of influenza B. New strains of influenza viruses can appear relatively frequently. Thus, the WHO and CDC recommends which strains to include in influenza vaccines and this can change each year. Currently, most approved influenza vaccines are produced in embryonated chicken eggs. These vaccines consist of inactivated whole or split subunit preparations. Production of these vaccines involves the adaptation of selected viral strains for high yield in eggs by either serial passage or reassortment with other high-yield strains. The selected influenza viruses are then mass-produced in chicken eggs, the progeny are purified from allantoic fluid and whole or split virus preparations are inactivated with a chemical agent such as formaldehyde. Egg-based vaccine production is time consuming, requires large biocontainment facilities, and relies upon the availability of millions of eggs. In the event of a pandemic outbreak, especially one involving a highly pathogenic avian influenza virus such as H5N1, these would be deadly limitations. Cell culture-based vaccine production using recombinant DNA technology, rather than live influenza virus, can overcome all of these limitations.
As mentioned above, the native HA molecule has covalently linked oligosaccharide side chains. Influenza HA's are classified as N-glycoproteins because their glycans are linked via amide (N), rather than glycosidic (O) bonds to the polypeptide chain. Different strains of influenza viruses can have variable numbers and types of N-glycans. The N-glycans at some sites on the HA polypeptide have high mannose structures, while those at others have a variety of more highly processed, complex structures (3, 7, 25, 27, 35, 37, 50). In fact, in a population of HA molecules, the same glycosylation site is typically occupied by a variety of different N-glycan species. One major type of complex N-glycan found on influenza HA's is a terminally galactosylated, biantennary structure with or without a bisecting N-acetylglucosamine residue, which also can have sulfated N-acetylglucosamine and galactose residues. Other types of complex N-glycans found on HA's are terminally galactosylated, tri- and tetraantennary structures.
In summary, existing vaccines are produced in eggs, which is a slow, unreliable, and potentially dangerous process. Clearly, alternative systems for influenza virus vaccine production are badly needed.